Previous Article | Next Article 
Journal of Virology, February 2000, p. 1794-1800, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The RNA Helicase and Nucleotide Triphosphatase
Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential
for Viral Replication
Baohua
Gu,
Changbao
Liu,
Juili
Lin-Goerke,
Derrick R.
Maley,
Lester L.
Gutshall,
Cynthia A.
Feltenberger, and
Alfred
M.
Del Vecchio*
Department of Molecular Virology and Host
Defense, SmithKline Beecham Pharmaceuticals, Collegeville,
Pennsylvania 19426-0989
Received 16 July 1999/Accepted 6 November 1999
Helicase/nucleoside triphosphatase (NTPase) motifs have been
identified in many RNA virus genomes. Similarly, all the members of the
Flaviviridae family contain conserved helicase/NTPase
motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it
is essential for virus replication is still unknown. To determine the
role of the NS3 helicase/NTPase in the viral life cycle, deletion and
point mutations in the helicase/NTPase motifs of the bovine viral
diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish
either helicase activity alone (motif II, DEYH to
DEYA) or both NTPase and helicase activity (motif I,
GKT to GAT and deletion of motif VI) were
generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362)
of these mutants and wild type was expressed in bacteria, purified, and
assayed for RNA helicase and ATPase activity. These mutations behaved
as predicted with respect to RNA helicase and NTPase activities in
vitro. When engineered back into an infectious cDNA for BVDV (NADL
strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that
these mutants did not synthesize minus-strand RNA. These findings
represent the first report unequivocably demonstrating that helicase
activity is essential for minus-strand synthesis.
*
Corresponding author. Mailing address: Department of
Molecular Virology and Host Defense, SmithKline Beecham
Pharmaceuticals, 1250 S. Collegeville Rd., P.O. Box 5089, Collegeville,
PA 19426-0989. Phone: (610) 917-6858. Fax: (610) 917-4170. E-mail:
alfred_m_delvecchio{at}sbphrd.com.
Journal of Virology, February 2000, p. 1794-1800, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Xiao, M., Wang, Y., Zhu, Z., Yu, J., Wan, L., Chen, J.
(2009). Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation. J. Gen. Virol.
90: 2923-2928
[Abstract]
[Full Text]
-
Xiao, M., Bai, Y., Xu, H., Geng, X., Chen, J., Wang, Y., Chen, J., Li, B.
(2008). Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation. J. Gen. Virol.
89: 994-999
[Abstract]
[Full Text]
-
Jeang, K.-T., Yedavalli, V.
(2006). Role of RNA helicases in HIV-1 replication. Nucleic Acids Res
34: 4198-4205
[Abstract]
[Full Text]
-
Wang, X., Lee, W.-M., Watanabe, T., Schwartz, M., Janda, M., Ahlquist, P.
(2005). Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates. J. Virol.
79: 13747-13758
[Abstract]
[Full Text]
-
Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H. M., Padmanabhan, R.
(2005). Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5'-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase. J. Biol. Chem.
280: 27412-27419
[Abstract]
[Full Text]
-
Lee, Y.-M., Tscherne, D. M., Yun, S.-I., Frolov, I., Rice, C. M.
(2005). Dual Mechanisms of Pestiviral Superinfection Exclusion at Entry and RNA Replication. J. Virol.
79: 3231-3242
[Abstract]
[Full Text]
-
Vlot, A. C., Laros, S. M., Bol, J. F.
(2003). Coordinate Replication of Alfalfa Mosaic Virus RNAs 1 and 2 Involves cis- and trans-Acting Functions of the Encoded Helicase-Like and Polymerase-Like Domains. J. Virol.
77: 10790-10798
[Abstract]
[Full Text]
-
Borowski, P., Lang, M., Haag, A., Schmitz, H., Choe, J., Chen, H.-M., Hosmane, R. S.
(2002). Characterization of Imidazo[4,5-d]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme. Antimicrob. Agents Chemother.
46: 1231-1239
[Abstract]
[Full Text]
-
Li, Y.-I., Shih, T.-W., Hsu, Y.-H., Han, Y.-T., Huang, Y.-L., Meng, M.
(2001). The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5' Cap Structure by Exhibiting RNA 5'-Triphosphatase Activity. J. Virol.
75: 12114-12120
[Abstract]
[Full Text]
-
Johnson, C. M., Perez, D. R., French, R., Merrick, W. C., Donis, R. O.
(2001). The NS5A protein of bovine viral diarrhoea virus interacts with the {alpha} subunit of translation elongation factor-1. J. Gen. Virol.
82: 2935-2943
[Abstract]
[Full Text]
-
Qu, L., McMullan, L. K., Rice, C. M.
(2001). Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity. J. Virol.
75: 10651-10662
[Abstract]
[Full Text]
-
Matusan, A. E., Pryor, M. J., Davidson, A. D., Wright, P. J.
(2001). Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication. J. Virol.
75: 9633-9643
[Abstract]
[Full Text]
-
Schweizer, M., Peterhans, E.
(2001). Noncytopathic Bovine Viral Diarrhea Virus Inhibits Double-Stranded RNA-Induced Apoptosis and Interferon Synthesis. J. Virol.
75: 4692-4698
[Abstract]
[Full Text]
-
Harada, T., Tautz, N., Thiel, H.-J.
(2000). E2-p7 Region of the Bovine Viral Diarrhea Virus Polyprotein: Processing and Functional Studies. J. Virol.
74: 9498-9506
[Abstract]
[Full Text]
-
Ahola, T., den Boon, J. A., Ahlquist, P.
(2000). Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping. J. Virol.
74: 8803-8811
[Abstract]
[Full Text]